ConSurfDB vs. ConSurf

Evolutionary Conservation is introduced in the article Conservation, Evolutionary, which also describes how conservation patterns in 3D can help to identify functional sites in proteins. Proteopedia displays conservation patterns pre-calculated by ConSurfDB. These are usually based on broad protein families that include sequences of proteins with multiple functions. Consequently, they usually obscure conservation present in a family of proteins with a single function (see Caveats). The present article describes the mechanisms utilized by the ConSurfDB and ConSurf servers, and how to use the latter to reveal conservation within a family of proteins with a single function.

The Two ConSurf Servers
There are two ConSurf servers:
 * ConSurfDB:
 * Has pre-calculated results for every chain in the PDB.
 * Proteopedia's Evolutionary Conservation resource displays results from ConSurfDB.
 * Results typically obscure some conservation related to a protein's function because the analysis typically included proteins of multiple functions (see ConSurfDB Often Obscures Some Functional Sites).


 * ConSurf:
 * You submit proteins of interest and wait for the analysis to be completed.
 * Enables you to pick the sequences used in the analysis from a list with checkboxes.
 * Highly flexible with many configurable parameters and several sequence database options.
 * You can upload your own multiple sequence alignment, or phylogenetic tree, for use in the analysis.

Both servers us state-of-the-art methods that are published in peer-reviewed journals. For comparisons with other methods, see Other Evolutionary Conservation Servers.

Both servers permit you to download results. This is a good idea since the continual growth of sequence databases and improvements in analysis algorithms will give at least slightly different results for the same jobs run several months or more apart. Also, results are periodically deleted from the ConSurf server to conserve disk space.

Examining Functions of Proteins in ConSurf-DB's MSA
As explained above, ConSurf-DB typically includes proteins with more than one function in its conservation analysis. Before deciding whether to do a ConSurf Server job that limits the analysis to proteins of a single function, you may want to see what proteins ConSurf-DB included in its analysis. Here is how to see the names (which hopefully reveal the functions) of the proteins included in ConSurf-DB's analysis of a protein chain. (The following steps are needed in May, 2009. A request to make this easier has been sent to the ConSurf-DB development team.)


 * 1) Go to consurfdb.tau.ac.il (the DB, distinct from the ConSurf Server).
 * 2) Enter the PDB code (PDB ID) for the protein of interest, and click the Show chains button.
 * 3) Click the button for complete, step by step computational results for the chain of interest.
 * 4) Under Alignment, note the number of sequences used.
 * 5) Under Output Files click on PSI-BLAST output. This will download the file seq.blast.zip.
 * 6) Windows XP or Vista:
 * 7) Double click on seq.blast.zip to unzip it. Right click on seq.blast and Copy. Right click on your Desktop (or elsewhere of your choosing) and Paste. Now you have the unzipped file seq.blast.
 * 8) Open seq.blast in a program that can number lines. (Notepad and Wordpad cannot number lines.) Start MS Word or the free Open Office Writer program (available from openoffice.org). Use the File menu to Open seq.blast.
 * 9) Delete everything above the first sequence, so the first sequence will be line number 1. The first sequence follows the header Sequences producing significant alignments:.
 * 10) Number the sequences by numbering the lines.
 * 11) MS Word: search for "add line numbers" to get instructions.
 * 12) Open Office Writer: Save the file as seq_blast.txt. (This enables line numbering.) Open the Tools menu, and select Line Numbering....
 * 13) Mac OS X:
 * 14) In the Finder, right-click (ctrl-click) on the file seq.blast, then Open With an application that can number lines of text. An excellent free one is Textwrangler from BareBones.Com.
 * 15) Delete everything above the first sequence, so the first sequence will be line number 1. The first sequence follows the header Sequences producing significant alignments:.
 * 16) Number the sequences by numbering the lines.
 * 17) MS Word: Set the Open dialog to enable All Files. Search for "add line numbers" to get instructions. You may need to select all and change the font (e.g. to Arial) to get the description of each sequence to fit on one line.
 * 18) TextWrangler (or BBEdit): Open the View menu, and under Text Display click Show Line Numbers.
 * 19) iWork Pages appears to lack a line numbering capability.
 * 20) Now you have the sequences numbered. Find the number equal to the number of sequences used reported under Alignment by ConSurf-DB.

If the functions of the proteins for this sequence number (and lower numbers) differ from that of the protein of interest, then ConSurf-DB included proteins of multiple functions in its analysis. This tends to obscure patches of conservation that exist among proteins with the same function as the query protein of interest.

Limiting ConSurf Analysis to Proteins of a Single Function
As explained above, the ConSurf-DB Evolutionary Conservation scene available in Proteopedia often includes proteins with multiple functions. However, the best way to find all functional sites by conservation analysis is to limit the analysis to proteins with a single function. A procedure for doing this follows. In June, 2009, the ConSurf development team is working on a new version that, once released, will enable selection of arbitrary sequences from the PSI-BLAST list.


 * 1) Go to consurf.tau.ac.il, the ConSurf Server (distinct from ConSurf-DB).
 * 2) If you are offered a choice between the new version and the old one, choose the new version.
 * 3) Fill out the form. When you get to the section Choose parameters to build the Multiple Sequence Alignment (MSA), be sure to check Let me select the sequences for the analysis manually out of Blast results.
 * 4) Under Maximum Homologs to collect, change 50 to all. The other options in this section can be left at their default settings.
 * 5) Enter your email address and click the Submit button.
 * 6) After a few minutes, a red message will appear "Please choose which sequences you want to use for ConSurf calculation". However, this message may appear low on the page, out of view, so scroll down periodically to check for it. The job cannot continue until you select the sequences.
 * 7) Scan the names of the proteins in the list that has checkboxes, under the header "Sequences producing significant alignments:". Find the first case where the function of the protein is not the same as the protein of interest (or the function is unclear). Usually you will want to exclude sequences for proteins of different functions.
 * 8) Scroll to the bottom of the page and put the number of the last sequence having the same function as the protein of interest in the box "Select the first [ .... ] sequences". Then click on the "Update selection" button.
 * 9) Scroll back to the top and make sure that only the desired sequences are checked. (Of course you may check or uncheck individual sequences if you wish.)
 * 10) When you are satisfied, scroll back to the bottom of the sequences list and click the Submit button.

The results of this "one function" job will usually enable you to identify more functional sites than did the ConSurf-DB result built into Proteopedia.

See below for instructions on how to make a green-link scene in Proteopedia that shows your single-function ConSurf result.

If your results have more than a few amino acids with insufficient data (yellow color ), you need more sequences. Repeat the procedure above with one change in the ConSurf job submission form: under Advanced Options, use the much larger Clean Uniprot database instead of the default Swiss-Prot database.

The ConSurf-DB Mechanism
Because results from the ConSurf DataBase server, ConSurf-DB are displayed within Proteopedia as Evolutionary Conservation, an overview of its methods is provided here. ConSurf-DB pre-calculates conservation levels for each amino acid in every protein chain in the Protein Data Bank. It went into service in 2008. It uses state-of-the-art methods, all published in peer-reviewed journals.

ConSurf-DB Process
An Average Pairwise Distance (APD) is calculated to describe the diversity of sequences in the MSA (see below). 
 * 1) A list of unique protein chains is extracted from the Protein Data Bank. Chains shorter than 30 amino acids are not processed because they do not contain enough information for reliable phylogenetic tree construction. Non-standard residues are converted to the closest standard amino acids. Chains with more than 15% non-standard residues are not processed. Chains that could not be processed are colored gray in Proteopedia -- see the color key at the top of this page.
 * 2) The amino acid sequence of each protein chain is submitted to PSI-BLAST for collection of related sequences from UniprotKB/Swiss-Prot . Three iterations are performed using an expectation value cutoff of 10-3.
 * 3) The sequences gathered with PSI-BLAST are then filtered (see below) using a scheme that attempts a balance between limiting the sequences to close homologues, and including distant sequences that do not share structure or function.
 * 4) The filtered sequence set is multiply aligned with MUSCLE (a multiple sequence alignment algorithm that out-performs CLUSTALW).
 * 5) A phylogenetic tree is constructed from the multiple sequence alignment (MSA) using the Rate4Site program developed by the ConSurf team.
 * 6) Rate4Site then calculates an evolutionary rate for each position in the MSA using a Bayesian approach shown by the ConSurf team to be superior . "The amino acid evolution is traced using the JTT substitution model. High evolutionary rate represents a variable position while low rate represents an evolutionarily conserved position."
 * 7) "The conservation scores are normalized so that the average over all residues is zero, and the standard deviation is one." Thus, conservation scores are relative, not absolute and comparing them between different protein families might be misleading (see Caveat above).
 * 8) The normalized conservation scores are then divided into nine levels from 1 (highly variable) to 9 (highly conserved).
 * 9) Colors mapped to the nine conservation levels, from turquoise (1) to burgandy (9) are applied to the 3D protein structure visualized in FirstGlance in Jmol. A coloring script for RasMol is also provided.
 * 1) A confidence interval for the conservation level is calculated for each amino acid position in the MSA. When this indicates low reliability, the position is colored yellow, signifying that the data were insufficient to assign a meaningful conservation level.

The results of each stage of the above process may be viewed for each chain at ConSurf-DB. In the initial run (February 2008), roughly 100 computer CPU's were utilized concurrently via a distributed computing system. Processing of the 30,918 unique protein chains in the PDB took about five days, or an average of roughly 30 minutes per chain.

Filtering
Filtering of the sequences gathered for each protein chain is crucial to making the ConSurfDB results maximally informative. Filtering consists of the following steps.
 * 1) Sequences with more than 95% sequence identity to the query sequence are discarded.
 * 2) Sequences shorter than 60% of the query sequence are discarded.
 * 3) Locally aligned sequence fragments that overlap by over 10% are discarded.
 * 4) Redundant sequences (>95% identical) are removed using CD-HIT.
 * 5) A maximum of 300 sequences meeting the above criteria is used (the 300 with the lowest expectation values, that is, most closely related to the query sequence).
 * 6) If the above process yields fewer than 50 sequences, the entire process is repeated using the Clean_UniProt database, which is about ten times larger than UniProtKB/Swiss-Prot. Clean_UniProt is a version of the UniProt database that attempts to exclude mutant or dubious sequences.
 * 7) If the above process yields fewer than 5 sequence homologs, no calculation is performed due to insufficient data. In February, 2008, this occurred for 1,348 chains out of 30,918 (4%).

Average Pairwise Distance
An Average Pairwise Distance (APD) is calculated to describe the diversity of sequences in the MSA generated during the processing of each chain. A value of 0.01 means that on average, there is one amino acid replacement for every 100 positions. Optimally informative results are obtained when the APD is between roughly 0.5 and 1.5.

The ConSurf Server
The ConSurf Server, first available in 2001  with many subsequent enhancements, can calculate and display the conservation pattern for 3D structures completely automatically. It should be used whenever the pre-calculated result at the ConSurf-DB needs improvement (for example, see above), or if you have your own multiple sequence alignment (MSA) that you wish to use. The default settings of ConSurf need to be adjusted in order to get an optimally informative result. For an example with default settings, see the cytochrome c comparision at ConSurf-DB. The main adjustment needed is to gather an adequate number of sequences for proteins of the same function as your protein of interest (see above).

Like ConSurf-DB, the ConSurf Server uses the same state-of-the-art methods, all of which are published in peer-reviewed journal articles. Unlike ConSurf-DB's pre-calculated results the ConSurf Server permits considerable customization. For example, the user may specify the number of sequences to use, choose the database from which sequences are obtained (Swiss-Prot or UniProt), set the Expectation cutoff, set the number of PSI-BLAST iterations, or submit their own multiple sequence alignment, or phylogenetic tree. Also you can upload your own PDB file, which enables you to process unpublished data, theoretical models, or "trimmed" chains, e.g. a domain of interest from a long chain.

In brief, the ConSurf Server uses the following process by default:
 * 1) Obtains the protein sequence for the specified PDB code (or uploaded PDB file) and chain.
 * 2) Gathers closely related sequences from Swiss-Prot (or a larger database that you specify) with a PSI-BLAST search. E value cutoff, number of iterations, and number of sequences to use are configurable.
 * 3) Filters the sequences, by default eliminating those redundant at 95% or higher identity with each other, and those with less than 35% sequence identity to the query sequence. These percentages are adjustable.
 * 4) Optionally enables the user to manually select which sequences will be used, from a list with checkboxes. In particular, this enables users to limit the analysis to proteins having the same function as the protein of interest (see above).
 * 5) Does a multiple sequence alignment with MAFFT. (Or you can choose a different algorithm or upload your own MSA.)
 * 6) Constructs a phylogenetic tree using neighbor joining with ML distance. (Or you can choose a different algorithm or upload your own tree.)
 * 7) Calculates a conservation score with confidence interval for each amino acid. Classifies the conservation scores into nine levels, and maps them to standard conservation level colors (see color key at the top of this page). Marks residues for which the conservation score confidence interval is too large, hence the conservation score is unreliable ("insufficient data").
 * 8) Displays the protein, colored by conservation, in interactive 3D, using FirstGlance in Jmol, Chimera, PyMOL, or Protein Explorer.

Examples
This example needs to be updated. It is on my list to do. Eric Martz 09:13, 25 April 2010 (IDT)

 At right is the pattern of evolutionary conservation and variability reported by ConSurf-DB for the alpha chain of Major Histocompatibility Complex Class I (chain A of 2vaa).

2vaa contains three chains. Here, ConSurf colors are applied only to the alpha chain (chain A), while the beta chain (chain B) and the peptide (chain P) are shown as gray backbone traces. Below are instructions for how to insert a ConSurf result into a Proteopedia scene.

Examples of conserved patches on other proteins, revealed by ConSurf, will be found in the articles on
 * Lac repressor
 * Avian Influenza Neuraminidase, Tamiflu and Relenza
 * Mechanosensitive channels: opening and closing